Eukaryotic cells may use the secretory pathway of plasmalemma assembly. Several membrane enzymes have been traced from their site of synthesis on endoplasmic reticulum-bound ribosomes through smooth ER, Golgi, and finally to the cell surface. The description of this pathway has been achieved by cytologic and cell fractionation analysis. Resolution at the molecular level will require genetic and biochemical techniques. This proposal deals with the application of genetic techniques to the dissection of the secretory-membrane growth process in saccharomyces cerevisiae. The available cytologic evidence suggests that yeast cells use secretory structures common to all eukaryotes. The potential for genetic analysis makes yeast an ideal experimental system. Conditionally lethal mutations which interrupt the secretory pathway should also block plasmalemma growth at a nonpermissive temperature, while not affecting soluble protein synthesis. Certain mutants affecting this pathway will accumulate secretory enzymes and organelles at a step prior to the defective one. Several mutants with these characteristics have recently been isolated in my laboratory. I propose to select a large number of conditional growth and secretion defective yeast mutants. Cytologic and histochemical techniques will be used to arrange a sequence of mutationally affected events. Genetic techniques will be used to confirm and extend the temporal position of mutant gene functions. The accumulated form of the secretory enzyme, invertase, will be isolated from various mutants, and its covalent structure will be examined. Finally, the contribution of the secretory pathway to plasmalemma growth will be evaluated by examining the fate of newly-synthesized polypeptides and lipids during incubation of secretory mutant at permissive and nonpermissive temperatures.